The purpose of this study is to engineer human T lymphocytes to be resistant to the near ubiquitous hypoxia and reactive oxygen species in the tumor microenvironment that impede the success of tumor immunotherapy. Aggressive hypoxic tumors are radio- and chemo-resistant, and metastasize rapidly. Hypoxia is detrimental to T cell viability and function, impeding anti-tumor immune response. Most tumors also constitutively express large quantities of reactive oxygen species (ROS), including hydrogen peroxide (H2O2), that promote their proliferation and further inhibit immune responses. Importantly, ROS production by is greatest in hypoxic tumors, with severe effects on immune effector mechanisms. Although adoptive transfer of cytotoxic T lymphocytes (CTLs) is a promising therapy for many cancers, they often fail to eliminate tumors or to provide long-term protection. We hypothesize that: 1) Low oxygen tension impairs the production of survival and growth promoting cytokines by CTLs and therefore their survival, proliferation, and differentiation into memory cells, and that these effects can be reversed by transgenic expression of the cytokine IL-15. 2) H2O2 induced immunosuppression can be overcome by overexpressing antioxidant proteins in T cells. 3) Such protected T cells can be redirected to additional tumor targets by forced expression of chimeric antigen receptors, reducing tumor escape by antigen loss and broadening the applicability of the engineered cells to a multiplicity of cancers. These hypotheses will be explored in three specific aims. In Aim 1, we will induce IL-15 expression by using transgenic sequences driven by a hypoxia-responsive element (HRE). Although our preliminary data show that such an approach using CTLs expressing HRE-IL-2 have improved survival and anti-tumor activity in hypoxic tumors, preliminary data also suggest that IL-15 will be even more potent and associated with lower toxicity. Aim 1 will test this proposition, comparing our results with HRE-IL-15 to those we obtained with HRE-IL-2. The most active of the IL-2/IL-15 cytokine-modified CTLs (CK-CTLs) will be used in Aims 2 and 3. In Aim 2, we will force CK-CTLs to overexpress NAD(P)H quinone oxidoreductase-1 (NQO1), an antioxidant that plays a dominant role in the upregulation of anti-apoptotic genes and protects T cells from ROS. We will investigate the ability of NQO1 to sustain CK-CTL expansion and favor survival in an environment rich in ROS. Our preliminary data show that NQO1-expressing CTLs can indeed survive and proliferate after H2O2 exposure. In Aim 3, we will further engineer these cytokine and NQO1 expressing CTLs to take advantage of their oxygen independence (OI-CTLs) and express a chimeric antigen receptor (CAR) directed to additional tumor-associated antigens. We will test our modified cells in a B-lymphoma xenograft model, and discover the safety, persistence, and anti-tumor activity of these modified T cells. Data obtained will be use as the sequential basis for separate clinical proposals in patients with advanced lymphoma, taking advantage of the exceptional translational research facilities of our institution.